Running mammalian cell culture that other people can reproduce: authenticating the line before it decides anything, choosing between serum and a defined medium, and the banking and mycoplasma testing that keep a culture trustworthy

The practices that decide whether the work is reproducible

1. Authenticate the line, then authenticate it again. Short tandem repeat profiling identifies a human line against a reference database and is inexpensive. Do it when the line arrives, before any publication and after any incident, because misidentified and cross-contaminated lines remain a substantial fraction of what circulates between laboratories.
2. Test for mycoplasma on a schedule, not on suspicion. Mycoplasma does not make the medium cloudy and changes gene expression, growth and drug response profoundly. Test at least quarterly and on every new arrival, quarantine incoming lines until they pass, and treat a positive as a reason to discard rather than to treat.
3. Bank in two tiers and work from the second. A master bank frozen at low passage and a working bank expanded from it. Everything for the study comes from the working bank, and when it is exhausted a new one is made from the master. Working continuously from a thawed vial guarantees drift nobody can measure.
4. Decide serum against a defined medium deliberately. Serum is undefined, varies between lots and carries supply risk; it also supports a great many lines that a defined medium does not. If you use it, test and reserve a lot. If you can move to a defined or serum-free formulation, the reproducibility gain is real and the adaptation takes weeks.
5. Record passage number with every experiment. Cells change with passage, and results from passage eight and passage forty are not comparable. Recording it costs nothing and is the single most useful number in a cell culture record.

Aseptic technique and the incubator

Most contamination arrives on hands and on bottles, not through the air. Work in a cabinet that has been certified, decontaminate everything entering it, and do not share a bottle of medium between people or between lines.

Clean and check the incubator on a schedule, including the water tray, and do not run antibiotics continuously. Routine antibiotics mask poor technique and select for resistant contamination that appears later and worse.

Freezing and thawing without losing the line

Freeze at high viability in the medium the line grows in with a cryoprotectant, cool slowly, and move to vapour phase nitrogen for long term storage. Thaw quickly and dilute out the cryoprotectant promptly.

Record the vial position, the passage and the viability at freezing. A bank nobody can navigate is a bank that gets thawed from the top, which is not the same as being used systematically.

What to write in a methods section

The line's source and catalogue identifier, the authentication method and date, the mycoplasma status, the medium and supplements with the serum lot where relevant, and the passage range used. That paragraph is what lets somebody else repeat the work.

Where a line was modified, state how and give the clone identity. A named parental line and an unnamed derivative is the most common gap in an otherwise complete methods section.

Common questions

How often should mammalian cell culture be tested for mycoplasma?

At least quarterly for lines in continuous use, and on arrival for every new line before it enters the main incubator. Infection is invisible without a test and changes results in ways that are easy to publish and hard to retract.

Do I really need to authenticate a line I got from a colleague?

Especially then. Lines passed between laboratories are the ones most likely to be misidentified, and a short tandem repeat profile is cheap compared with a study built on the wrong cells.

Serum or serum-free medium?

Serum-free where the line supports it, for reproducibility, defined composition and supply security. Serum where the line needs it, in which case test candidate lots against your own assay and reserve the one that works.

How many passages is too many?

It depends on the line, and the way to know is to measure: growth rate, morphology and the assay you care about, at intervals. Set a maximum passage from that evidence and return to the working bank when it is reached.

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